The interaction of proteins with each other and with other cellular components is an intrinsic part of nearly every cellular process, and this is especially true of intracellular signalling systems. Information is passed through and between signalling systems by a series of such interactions. In order to study the function of a protein, a practical strategy is first to identify the components that it interacts with. Most of these will be other proteins—sometimes of the same species, but most often a very different type and with very different functional characteristics.
The identification of novel interactions is a very rapidly growing area of research in cell biology and signal transduction. A notable feature of recent discoveries in this area is the specificity with which partners interact, equalling or exceeding the degree of specificity commonly seen in ligand-receptor interactions seen at the cell surface. Identification of interacting species brings with it the opportunity to identify novel signalling interactions that may assist greatly in the functional characterisabon of proteins involved in cellular signalling. In addition the method will be applicable to the development of new pharmaceutical agents capable of disrupting or engaging partners in an interaction.
Compounds with this mechanism of action will be able to modulate the flow of information through signalling pathways, and in so doing find application in very many areas of human and animal health care. Since such compounds will be inherently very selective and have their action without the need for gross inhibition of catalytic activity, it can be expected that therapies based on their use will not carry with them the problems of poor specificity and damaging side effects commonly associated with more traditional active-site inhibitors.
Existing methods for identification of interacting species can be divided into two groups: First are those methods that can only work with more or less purified components brought together in vitro, such as surface plasmon resonance (evanescent wave methods), protein mass spectroscopy, fluorescence correlation spectroscopy and anisotropy measurements all with the common feature that the components of interest are isolated from the cellular context. The second group includes all methods designed to work within living cells. Of these, many have been developed to work in yeast cells (yeast two hybrid, reverse yeast two hybrid and variations thereof) but some have been adapted for use in mammalian cell systems. Cellular methods for detection of protein interactions have been well reviewed by Mendelsohn, A. R., Brent, R. (1999) (Science 284(5422):1948). Many of these methods are descendants of the conventional two-hybrid methods, where transcriptional activity is initiated by the bringing together of bi-partite transcription factors through the interaction of attached “bait” and “prey” components, while other methods rely on reconstitution of a biochemical function in vivo. Rossi et al. (2000) (Trends in Cell Biology 10:119-122) have thus developed a mammalian cell-based protein-protein interaction assay where the read-out is not transcriptional but reconstitution of a mutated beta-galactosidase enzyme. Upon reconstitution of the tetrameric enzyme, enzymatic activity can be monitored. In addition methods for monitoring protein-protein interactions that are based on an optical read-out i.e. fluorescence resonance transfer (FRET), or coincidence analysis (a variant of fluorescence correlation spectroscopy), or fluorescence lifetime changes. The last three categories are more normally applied under simplified in vitro conditions, but attempts are being made to move them into the more complex environment of the living cell.
Recently Tobias Meyer reported (WO00/17221) a method wherein two heterologous conjugates are introduced into a cell. The first heterologous conjugate comprises the first protein of interest conjugated to a detectable group (e.g. GFP). The second heterologous conjugate comprises a second protein of interest conjugated to a protein that specifically binds to an internal structure within the cell upon stimulation with phorbol ester. When the second protein is bound to an internal structure within the cell, with a known distribution, binding between the two proteins of interest can be visualised as the detectable group will be located bound to internal structure within the cell.
Proteins(-GFP) that are not “anchored” in an intracellular organelle or compartment, but are more or less mobile in the cytosol, will diffuse into the surrounding medium upon cell permeabilization, at a rate largely governed by the degree of permeabilisation imposed. It is difficult to control the release of cellular contents by permeabilization since detergents have the tendency to not only disrupt the membrane but also over time disrupt intracellular components. Beside this, damaging the membrane will start some uncontrolled protease activity with the same unintentional result. This phenomenon is also seen when using non detergent permeabilization (eg. Digitonin). Fixative agents are commonly used to preserve structural integrity in cells during the processes necessary to prepare biological material for microscopy. Fixatives aimed at preserving or stabilising protein structures within cells can be divided into two groups; those that coagulate proteins, such as organic acids or alcohols (e.g. acetic acid, ethanol), and those that cross-link proteins together into an insoluble network, such as the  aldehydes (e.g. formaldehyde or glutaraldehyde). The rate of fixation by such agents is governed by their rate of penetrance into cells together with the rate of chemical cross-linking or coagulation that they can achieve. The processes and methods of cellular fixation have been thoroughly studied and described in the scientific literature (see, for example, Fixation for Electron Microscopy by M. A. Hayat, 1981, Academic Press, New York).
Translocation usually involves changes in the effective mobility of at least one protein within the cell, either through changes in interaction between that component and an anchored (or effectively non-mobile) component or through a change in location or compartmentation of the component, for example a transfer from the extra-nuclear cytoplasm into the nucleus itself. Changes in interaction or compartmentation constitute translocation. Redistribution™ is the art of making translocation a measurable event (WO98/45704). Translocation may involve change in mobility or compartmentation of a component to which the component of interest is attached, for example a motor protein by which the component of interest is carried. Some translocations involve sub-microscopic distances, for instance the interaction between a soluble signaling protein in the cytoplasm and an adjacent actin filament or intracellular membrane. The mobility of the previously soluble component is greatly changed in such a translocation, but any change in its position in space may be unresolvable by microscopic means. In this case, it would be desirable to separate mobile from immobile forms of the protein of interest in order to reveal the redistribution signal.
Usually redistribution™ is measured by “imaging”, which is very time consuming and inconvenient for drug screening in HT format. Redistribution™ assays often comprise cell lines stably expressing a particular protein of interest, (most often that protein being an engineered protein fusion between the protein of interest and a luminescent protein such as GFP). In such cells, it may be the case that the component of interest is overexpressed to some degree relative to the other components in the cell with which it should interact. This can lead to a masking of the translocation event by excess amounts of the protein of interest that cannot physically interact with the limited number of partner components available in the cell. In such cases, removal of the excess component of interest may be sufficient to unmask the translocation that has occurred.
Fixation of the cells before permeabilization is never successful in such cases, since all proteins become effectively immobilized to approximately the same degree under the influence of the fixative, and hence cannot be differentially removed or washed away from the cell.